The determination of the amino acid sequence of a peptide is essential to understanding its structure, as well as to modifying the peptide to achieve desired properties in an analog or mimetic. The most widely used method of peptide sequencing involves reacting at the N-terminus with phenyl isothiocyanate (PITC), a process known as Edman degradation (Edman). The reaction of PITC with the terminal amino group adds a phenylthiourea group, which cyclizes and cleaves, forming a free anilinothiozolanone (ATZ) of the N-terminal amino acid, and a shortened peptide. The ATZ-derivative of the N-terminal amino acid is separated, converted to the corresponding phenylthiohydantoin (PTH), and identified by HPLC.
N-terminal sequencing is carried out by successively converting the next-in N-terminal amino acid to the free amino acid PTH, and identifying each successively released amino acid. The method is generally reliable for sequences up to about 20-40 amino acid residues and is readily performed with automated instrumentation. Of course, for longer polypeptides, C-terminal sequence information is not available by N-terminal sequencing.
Several methods have been proposed for C-terminal peptide sequencing, based in methods which are primarily enzymatic, physical, or chemical. The enzymatic strategy involves analyzing the amino acids released from treatment of the peptide with carboxypeptidases. It is impeded by difficulties in controlling the extent, the rate and the specificity of enzymatic cleavages.
The most common physical tools used for C-terminal sequencing are fast atom bombardment mass spectrometry (FAB/MS), and nuclear magnetic resonance (NMR) spectroscopy. FAB/MS analysis is applicable to 1-10 nmole amounts of peptide, but requires expensive mass spectrometry equipment. Sequence determination by NMR utilizes large amounts of peptide, typically in the .mu.molar range, and also involves expensive equipment.
In view of the limitations of enzymatic and physical approaches to C-terminal sequencing, considerable effort has been invested in developing chemical methods for determining C-terminal amino acids residues, and for C-terminal sequencing. An inherent difficulty in C-terminal sequencing is the relative chemical inertness of the carboxyl/carboxylate group, in contrast to the reactivity of the amino group.
Several chemical methods have been proposed for C-terminal sequencing (Inglis). One of these involves generating a carboxyamido derivative at the C-terminal end of the peptide, followed by derivitization with bis(I,I-trifluoro-acetoxy)iodobenzene, and hydrolysis to form a shortened carboxyamidopeptide and the aldehyde derivative of the C-terminal amino acid (Parham). A second and related approach involves reacting the carboxyl terminus with pivaloylhydroxamide to effect a Lossen rearrangement. One limitation of this method is that it does not progress beyond aspartic or glutamic acid residues (Miller, 1977).
The most widely studied of the C-terminal sequencing chemistries is the thiohydantoin (TH) reaction. In one general procedure for carrying out the thiohydantoin method, the carboxyl group is activated with an anhydride, in the presence of an isothiocyanate (ITC) salt or acid, to form a C-terminal peptidylthiohydantoin via a C-terminal ITC intermediate (Stark). The thiohydantoin is a ring formation which includes the nitrogen, chiral carbon and carbonyl of the terminal amino acid. It is attached to the remainder of the peptide through the bond which was the amide bond between the C-terminal and penultimate amino acids. The C-terminal thiohydantoin can be cleaved at that amide bond, producing a shortened peptide and the thiohydantoin derivative of the C-terminal amino acid. This derivative can be separated and identified, e.g., by high performance liquid chromatography (HPLC).
However, the chemical reactions used in thiohydantoin-based methods present two significant disadvantages when applied in amino acid sequencing. The initial formation of the C-terminal amino acid thiohydantoin typically requires long reaction times with highly reactive agents, such as anhydrides. (Meuth, Shively et al.). Such conditions often lead to chemical alterations of the peptide. The second drawback encountered with TH reactions is the difficulty in efficiently cleaving the C-terminal amino acid thiohydantoin from the peptide. Since the thiohydantoin group is not a good leaving group, strong reagents (e.g., a strong acid, base or nucleophile) and fairly vigorous conditions are required to achieve efficient cleavage, again compromising the integrity of the remaining peptide.
Recent efforts for improving thiohydantoin chemistry have focused on improving the efficiency of the C-terminal amino acid thiohydantoin formation. For example, Boyd et al., U.S. Ser. No. 07/546,303 discloses improved methods of C-terminal amino acid thiohydantoin formation which can be carried out under milder conditions. Hawke et al., U.S. Ser. No. 07/454,666 and U.S. Ser. No. 07/457,088, also discloses improved methods for thiohydantoin formation. The disclosures of each of these references is incorporated herein by reference in their entirety.